PROJECT SUMMARY The overall goal of this research is to increase our understanding of how cilia beat by dissecting the molecular mechanisms and regulation of ciliary motility on a molecular level. Cilia and flagella are conserved and ubiquitous microtubule-based organelles with important roles in cell locomotion, fluid transport, sensation, cell signaling and development, which are critical processes for the survival and proper function of many eukaryotic cells and tissues. In humans, defects in the motility and assembly of cilia are responsible for numerous congenital diseases, such as primary ciliary dyskinesia, chronic respiratory disease, impaired fertility, brain developmental defects, congenital heart disease and randomization of the left-right body axis. Cilia motility is driven by the coordinated activities of thousands of dynein molecules, comprised of multiple isoforms. Our previous studies of wild type and mutant cilia, and actively beating cilia have opened a new window into the functional organization of motile cilia. However, long-standing fundamental questions remain about how regulatory signals change dynein?s activity on a molecular level, what are the roles of the different regulatory complexes during ciliary motility, and how dyneins are spatially and temporally coordinated to generate the oscillatory beating typical for cilia. Building on a strong premise of both published and preliminary new data, this proposal directly addresses these critical gaps through three specific aims that are directed at (Aim 1) revealing mechanisms by which dynein?s action is regulated to initiate and propagate ciliary waves, (Aim 2) determine the patterns of dynein activity that generate different ciliary waveforms, and (Aim 3) characterizing ciliary components that assemble only on specific doublets to ask if their inherently asymmetric distribution contributes to producing ciliary beating. We use a powerful and innovative combination of modern approaches that include cryo-electron tomography to image mutant cilia and tagged proteins with molecular resolution, genetics and proteomics, an alternate model organism to study cilia, and a state-of-the-art ?cutting? technique to look ?deeper inside? cells than previously possible. We expect that our combined studies will provide important new conceptual and mechanistic insights into ciliary motility and regulation, which will also impact our understanding of ciliary diseases.